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This paper describes the preparation of pyridine derivatives of 2,3-dichloro-5(8?)-nitro-1,4-naphthoquinone. A method for the nitration of 2,3-dichloro-1,4-naphthoquinone is also described. Certain 4-n-alkyl, 3,4-dialkyl, and 4-cycloalkyl pyridine derivatives are caused to undergo condensation reactions with the nitrated naphthoquinone, giving rise to a series of compounds of the preceding type (X). All of the compounds prepared will be tested for anti-tubercular activity by Parke-Davis and Company.

Bakgrund Förmaksflimmer är en arytmi som uppkommer av att sinusknutan slutar styra hjärtrytmen och impulser initieras istället på flera olika ställen i förmaken. Detta orsakar en snabb och oregelbunden kontraktion med försämrad cirkulation som resultat. Förmaksflimmer är den vanligaste orsaken till stroke och en viktig del i behandlingen av förmaksflimmer är därför att förebygga stroke genom antikoagulerande läkemedel. Warfarin har länge varit förstahandsval, men nya läkemedel, så kallade icke vitamin K antagonist oral antikoagulantia (NOAK) har de senaste åren godkänts som förebyggande behandling vid indikationen förmaksflimmer. Syftet med arbetet var att undersöka effekt, blödningsrisk och kostnad av NOAK som förebyggande behandling av stroke och systemisk emboli hos patienter med förmaksflimmer.  Metod och material Arbetet utfördes som en litteraturstudie där fem kliniska studier från databasen PubMed analyserades. I fyra studier jämfördes de tre faktor Xa-hämmarna apixaban, edoxaban och rivaroxaban, samt trombinhämmaren dabigatran med warfarin. I en studie jämfördes apixaban med Aspirin®.  Resultat Alla NOAK visades reducera risken att drabbas av stroke och emboli minst likvärdigt med warfarin. Dabigatran 150 mg och edoxaban 60 mg visades även vara effektivare än warfarin (RR=0,66; P<0,001, respektive RK=0,79; P<0,001). Apixaban reducerade risken för stroke och systemisk emboli med mer än 50 % i jämförelse med Aspirin® (RK= 0,45; P<0,001). Uppkomst av större blödning var likvärdigt förekommande i jämförelse mellan NOAK och warfarin. Dabigatran 110 mg, edoxaban 30 mg, edoxaban 60 mg och apixaban 5 mg visade på lägre risk för större blödning. Apixaban och Aspirin® visades vara likvärdiga avseende uppkomst av större blödning. Slutsats Icke vitamin K antagonist oral antikoagulantia är effektiva som förebyggande behandling av stroke och emboli hos patienter med förmaksflimmer, med lägre blödningsrisk än warfarin, men till en högre kostnad. / Atrial fibrillation is an arrhythmia characterized by rapid and uncontrolled contraction of the atria. The irregular contractions leads to incomplete circulation, accumulation of blood in the atria and increases the risk of stroke and embolism. An important part in the treatment of atrial fibrillation is to prevent the risk of stroke by use of anticoagulants. The first line treatment is the vitamin K antagonist warfarin. The drug has many side effects such as risk of bleeding, difficulties to adjust the dose and interactions with both drugs and food. In recent years, new drugs, called non vitamin K antagonist oral anticoagulants (NOAC), have been approved as preventive treatment of stroke in patients with atrial fibrillation. These include three factor Xa inhibitors: apixaban, rivaroxaban, and edoxaban, and one thrombin inhibitor: dabigatran. In this study, the efficacy, risk of bleeding and cost of NOAK was investigated for the prevention of stroke and systemic embolism in patients with atrial fibrillation. The study was conducted as a literature study where five clinical trials from the database PubMed was analyzed. In four studies, the three factor Xa inhibitors apixaban, edoxaban and rivaroxaban, and the thrombin inhibitor dabigatran were compared with warfarin. In one study apixaban was compared with Aspirin®. In all studies the prevention of stroke and systemic embolism and risk of bleeding was investigated. All NOAC reduced the risk of stroke and embolism at least equal to warfarin. Dabigatran 150 mg and edoxaban 60 mg was also more effective than warfarin. Apixaban reduced the risk of stroke and systemic embolism with more than 50 % compared with aspirin. The occurrence of major bleeding was similar in comparison of Dabigatran 150 mg, respectively rivaroxaban 20 mg and warfarin. Dabigatran 110 mg, edoxaban 30 mg, edoxaban 60 mg and apixaban 5 mg showed a lower risk of major bleeding than warfarin. Apixaban and Aspirin® appeared to be equivalent regarding the occurrence of major bleeding. Non Vitamin K antagonist oral anticoagulants are effective in the prevention of stroke and embolism in patients with atrial fibrillation, with lower risk of bleeding than warfarin, but with a higher cost.

This thesis describes the development of separate assays to measure coenzyme Q₁₀ (CoQ₁₀) and vitamin K. Coenzyme Q is essential for the mitochondrial electron transport chain, and vitamin K for the blood coagulation cascade. Vitamin K deficiency is associated with haemorrhagic disease of the new-born, and CoQ₁₀ deficiency with HMG-CoA-reductase inhibitor (statin) therapy and heart failure. Coenzyme Q and vitamin K are usually measured by HPLC, using electrochemical and ultraviolet, and electrochemical and fluorescence detection, respectively. For vitamin K1, the limit of detection achieved using fluorescence and electrochemical detection was 0.28 and 0.12 nmol/L, respectively. Sensitivity of fluorescence detection is improved by using protic solvents in the mobile phase, and platinum-black catalysed alcohol reduction. The lipophilicity and low endogenous concentrations of vitamin K1 hinder its measurement, and further work is required to produce a rapid, reliable and robust assay for its measurement in human plasma. The limits of detection achieved using fluorescence, ultraviolet and electrochemical detection to measure CoQ₁₀ were 29, 4.8, and 0.34 nmol/L, respectively. Plasma CoQ₁₀ is not stable during long term storage at -13 ℃, but at -80 ℃ it is stable for at least 18 months. The reference interval for plasma total CoQ₁₀ in the New Zealand population is 0.47 - 1.80 µmol/L. There is no clinical requirement for stratification of the reference interval according to gender. Coenzyme Q₁₀ in human plasma is homeostatically controlled, varying little over a two month interval in healthy young males. Coenzyme Q₁₀ supplements have significantly different bioavailability, with the median increase in plasma CoQ₁₀ ranging from 0.14 to 0.59 µmol/L for seven different supplement brands. There is a large inter-individual variation in CoQ₁₀ absorption, and hence plasma concentrations should be monitored during supplementation. A plateau in CoQ₁₀ absorption, from a single dose, at approximately 200 mg suggests that the maximum dose ingested at one time should be 200 mg or less. Q-Gel capsules containing 30 mg of CoQ₁₀ are twice as effective at raising blood CoQ₁₀ as 100 mg capsules. Plasma CoQ₁₀ in patients with chronic heart failure are significantly lowered by approximately 33% when these patients receive Atorvastatin for six weeks. The absolute decrease in CoQ₁₀ showed a significant correlation with worsening endothelial function (r = + 0.548, p = 0.011). Coenzyme Q₉ was shown to be present in human plasma with a reference interval of 8.8 - 47.0 nmol/L.

Seit der Entdeckung des ersten Gens für den Vitamin K Epoxid-Reduktase-Komplex (VKORC1), dem Schlüssel-Enzym für die Regenerierung von Vitamin K, sind keine zusätzlichen Komponenten des Komplexes beschrieben worden. Die einzige bekannte Funktion von VKORC1 ist bislang die Reduktion von Vitamin K-2,3-Epoxid, welches bei der post-translationalen Carboxylierung von Proteinen als oxidierter Kofaktor anfällt, und im sogenannten Vitamin K-Zyklus regeneriert wird. VKORC1 ist zugleich das Zielprotein antikoagulativer Medikamente der Coumarin-Gruppe, wie Warfarin oder Marcumar. Mutationen im VKORC1-Gen führen zu einem signifikanten Effekt auf die benötigte Coumarin-Dosis und die Stabilität der Hämostase in der Thrombosebehandlung mit Antikoagulanzien. Gleichzeitig mit VKORC1 wurde ein stark sequenz-homologes Protein identifiziert, das „VKORC1-like1“ (VKORC1L1) genannt wurde und dessen physiologische Funktion zu Beginn dieser Arbeit völlig unbekannt war. Die vorliegende Arbeit beschäftigte sich mit zwei Aspekten des Vitamin K-Stoffwechsels: A. Den enzym-kinetischen Eigenschaften und der subzellulären Lokalisation des VKORC1L1-Proteins sowie B. der Identifizierung und Charakterisierung von Proteinen, die Interaktionspartner der beiden VKOR-Proteine sein können. Die Ergebnisse können wie folgt zusammengefasst werden: A.1. Die enzym-kinetischen Untersuchungen zeigen starke Ähnlichkeiten zwischen VKORC1 und VKORC1L1: Beide Enzyme haben eine Vitamin K-Epoxidase- und -Reduktase-Aktivität, wobei die Affinitäten zu Vitamin K2-Epoxid deutlich höher sind als die zu Vitamin K1-Epoxid. Beide Enzyme sind durch Warfarin hemmbar und der Austausch der vermutlich am Elektronentransfer beteiligten Cysteine an homologen Positionen führt in beiden Proteinen zu einem fast vollständigen Verlust der Aktivität. A.2. Mittels Ko-Lokalisation konnte VKORC1L1 – wie VKORC1 – in der ER-Membran lokalisiert werden. Folglich schließen wir, dass VKORC1L1 eine ähnliche Struktur, die gleiche zelluläre Lokalisation und zumindest in vitro auch eine VKOR-Aktivität hat und daher eventuell eine weitere Komponente des VKOR-Komplexes darstellen könnte. Allerdings sprechen gewichtige Argumente dagegen, dass beide Proteine funktionell austauschbar sind: Sowohl bei Patienten mit Mutationen in VKORC1 (VKCDF2-Erkrankung), als auch bei VKORC1-Knock-out Mäusen kann das intaktes VKORC1L1-Protein die inaktivierende Mutation im C1-Gen nicht kompensieren. B.1. Mit einem für Membranproteine adaptierten, modifizierten Yeast-Two-Hybrid Screen (Split-Ubiquitin-System) konnten mit VKORC1 und VKORC1L1 als Köder 114 Proteine aus einer Leber-cDNA-Bank als potentielle Interaktionspartner identifiziert werden. Davon wurden 6 Proteine aufgrund ihrer Trefferhäufigkeit und funktioneller Überlegungen mit Hilfe von Ko-Immunpräzipitationsexperimenten und Ko-Immunlokalisation näher untersucht. Interessanterweise zeigen die beiden Trefferlisten starke Überschneidungen. B.2. Es konnte eine in vitro- Interaktion von VKORC1 mit sich selbst und mit VKORC1L1 nachgewiesen werden, die bisher nicht bekannt war. Dies könnte auf der hohen Sequenz- und Struktur-Homologie der beiden Proteine beruhen, führt aber auch zu neuen Hypothesen bezüglich des Vitamin K-Stoffwechsels. B.3. Die Interaktion von VKORC1 und dem „stress-associated endoplasmic reticulum protein 1“ (SERP1) bringt Vitamin K in Zusammenhang mit oxidativem Stress. Dazu passen auch die neuesten Ergebnisse aus der Arbeitsgruppe von Johannes Oldenburg (vormals Würzburg, jetzt Bonn) zur Funktion von VKORC1L1, die eine protektive Rolle von Vitamin K beim Schutz der Zelle vor reaktiven Sauerstoffverbindungen nahe legen. Ob und wie Vitamin K und VKORC1L1 einen neuen Schutzmechanismus gegen Sauerstoffradikale bilden bedarf weiterer Untersuchungen. B.4. Ferner wurde eine Interaktion zwischen VKORC1 und dem „Emopamil-binding-protein“ (EBP) nachgewiesen. Mutationen in EBP führen zu der seltenen genetischen Krankheit Chondrodysplasia punctata. Die Ähnlichkeit der Symptomatik zwischen Chondrodysplasia punctata und der sogenannten Warfarin-Embryopathie, die durch überhöhte Dosierung von Coumarinen während der Schwangerschaft verursacht wird, legt einen Zusammenhang zwischen Vitamin K- und dem Kalziumstoffwechsel nahe. B5. In den Ko-Immunpräzipitationsexperimenten nicht bestätigt haben sich die initial positiven Proteine Protein-Disulfid-Isomerase (PDIA6), CD63, und Fibrinogen-Gamma (FGG). Die Ergebnisse geben Hinweise auf neue Funktionen der VKOR-Proteine beim Schutz vor oxidativem Stress und in der Verbindung zum Kalzium-Stoffwechsel. Beide Aspekte bedürfen weiterführender Untersuchungen. Im Hinblick auf diese neuen Funktionen wäre auch eine kritische Betrachtung der übrigen 85 primären Treffer des Split-Ubiquitin-Screens sinnvoll. / Since the first gene of the vitamin K epoxide reductase complex (VKORC1) - the key enzyme for the regeneration of vitamin KH2 - has been discovered, no more components of the complex have been described. To date, the only known function of VKORC1 is the reduction of vitamin K-2,3-epoxide (VKO) in the vitamin K cycle. VKO plays a role as an oxidative cofactor during post-translation carboxylation. VKORC1 is also the target protein of anticoagulant drugs of the coumarin type (e.g. warfarin or marcumar). Genetic variants in VKORC1 have recently been shown to significantly affect the coumarin dose and the stability of hamostasis (measured as INR level) during thrombosis treatment. Simultaneously with VKORC1, a homologues protein called VKORC1-like1 (VKORC1L1) was identified. The physiological function of VKORC1L1 was unknown at the beginning of this thesis. The present thesis focuses on two aspects: A. Kinetic properties and subcellular localization of VKORC1L1-protein as well as B. identification and characterization of potential interaction partners of VKORC1 and VKORC1L1, respectively. The results could be summarised as follows: A.1. Kinetic studies revealed a high similarity of VKORC1 and VKORC1L1: Both VKOR-proteins have shown a VKR-activity in our assay and a higher affinity for Vitamin K2-epoxid than for Vitamin K1-epoxid. Both enzymes could be inhibited by warfarin and the exchange of homologous Cysteins leads to an almost complete loss of function for both proteins. A.2. Localisation studies identified VKORC1L1 in the ER when co-expressed in HeLa cells with VKORC1. Accordingly, we suggest that VKORC1L1 has a similar function, localisation and structure as VKORC1 and therefore becomes a potential component of the VKOR-complex. Nevertheless, important arguments lead to the fact that both proteins are not interchangeable as neither patients with VKCDF2 nor VKORC1-knock out mice could be counterbalanced by VKORC1L1. B. Using the split-ubiquitin-system, a method based on yeast two-hybrid system which is capable to investigate membrane-proteins, 114 potential interactions candidates for VKORC1 and VKORC1L1 were found in a human adult liver cDNA library screen. 5 of them were further investigated using co-immunprecipitation and co-localisation studies. Interestingly, various similar hits could be detected for both proteins. B.1. For the first time we were able to show an in vitro-interaction of VKORC1 with itself and the homologues protein VKORC1L1. This might be due to the high level of homology between the two proteins but also leads to a new hypothesis in respect to the vitamin K metabolism. B.2. The interaction of VKORC1 and “stress-associated endoplasmatic reticulum protein 1” (SERP1) connects vitamin K with oxidative stress. This is in accordance with new results from the group of J. Oldenburg (formerly Würzburg, now Bonn). They postulate an antioxidant role of VKORC1L1. Whether VKORC1L1 and vitamin K have an antioxidative effect needs to be solved in further studies. B.2. Furthermore, we showed an interaction between VKORC1 and the “emopamil binding protein” (EBP). Genetic variants in EBP lead to a rare genetic disease chondrodysplasia punctata which can be considered a phenocopy of warfarin embryopathy, which is caused by medication of woman with coumarin derivatives during the first trimester of pregnancy. This interaction and the similar phenotypes suggest a connection between the vitamin K- and calcium metabolism. B.3. The following proteins, which were found using split-ubiquitin-system, could not be confirmed via co-immunoprecipitaion: protein-disulfide-isomerase A6 (PDIA6), CD63, a member of the tetraspanin-family, and fibrinogen-gamma-chain (FGG). A PDI-form was proposed to provide electrons for reduction of the thioredoxin-like CXXC center in VKORC1. Tetraspanins are a large family of cell-surface proteins, which interact with proteins in a molecular network that is known as the tetraspanin web. FGG is the gamma component of fibrinogen, the only identified protein which is known to play a role in blood coagulation. These results suggest new functions of VKOR-proteins related to protection against oxidative stress and a connection to the calcium-metabolism. Both aspects need to be analysed further. Therefore, it would make sense to analyse the remaining candidates found via split-ubiquitin-system with regard to these new functions.

The role of vitamin K in the blood clotting cascade has been well documented. Vitamin K has recently been implicated in improving bone health. The current studies were conducted to determine the effects of vitamin K in diets with and without mycotoxin contaminated corn on growth performance, bone characteristics, and related blood metabolites in pigs from weaning to market. Menadione sodium bisulfite complex (MSBC, 33% vitamin K) was chosen as the source of supplemental vitamin K because it is the most common form fed to swine. Vitamin K was tested at 0, 0.5, and 2.0 ppm in a corn-soybean meal based diets on two generations of pigs to evaluate any time and dose responses. The first generation of pigs was subjected to mycotoxin contaminated corn in the nursery phase to test for any interactions between the toxins and vitamin K. The addition of 0.5 ppm vitamin K reduced (P < 0.0001) prothrombin time. No additional decrease in prothrombin time was detected when increasing vitamin K inclusion from 0.5 to 2.0 ppm. With regard to growth performance, daily gain, feed intake, and feed efficiency were unaffected (P > 0.10) by supplemental vitamin K. However, pigs fed mycotoxin contaminated corn ate less (P = 0.005) and grew slower (P = 0.015) compared to those receiving good corn. The addition of vitamin K did not alleviate the negative growth effects in response to corn type. Vitamin K did not affect bone characteristics (P > 0.10), blood Ca (P > 0.05) or OC (P > 0.10). Other than blood clotting it does not appear that dietary vitamin K provides any additional benefits at these levels of inclusion and stages of swine production.

The objective of this study was to investigate the extent to which vitamin D and K are associated with bone health in pediatric cystic fibrosis (CF) patients. We hypothesized that: (1) the prevalence of vitamin D and K deficiencies would be high despite routine vitamin therapy, (2) bone health would be reduced and (3) vitamin K and D status would be associated with bone health. / Our results showed poor bone mineral mass in these CF children despite mild disease and good nutritional status. Neither vitamin K nor D was a predictor of bone health but weight and height Z-scores, fat-free mass, physical activity and lung function were all consistent predictors. / These results indicate that nutritional status as well as physical activity are key determinants of bone health in CF children and offer a unique opportunity in the prevention of CF-related bone disease. Further vitamin intervention research needs to be done in this population.

This thesis describes the development of separate assays to measure coenzyme Q₁₀ (CoQ₁₀) and vitamin K. Coenzyme Q is essential for the mitochondrial electron transport chain, and vitamin K for the blood coagulation cascade. Vitamin K deficiency is associated with haemorrhagic disease of the new-born, and CoQ₁₀ deficiency with HMG-CoA-reductase inhibitor (statin) therapy and heart failure. Coenzyme Q and vitamin K are usually measured by HPLC, using electrochemical and ultraviolet, and electrochemical and fluorescence detection, respectively. For vitamin K1, the limit of detection achieved using fluorescence and electrochemical detection was 0.28 and 0.12 nmol/L, respectively. Sensitivity of fluorescence detection is improved by using protic solvents in the mobile phase, and platinum-black catalysed alcohol reduction. The lipophilicity and low endogenous concentrations of vitamin K1 hinder its measurement, and further work is required to produce a rapid, reliable and robust assay for its measurement in human plasma. The limits of detection achieved using fluorescence, ultraviolet and electrochemical detection to measure CoQ₁₀ were 29, 4.8, and 0.34 nmol/L, respectively. Plasma CoQ₁₀ is not stable during long term storage at -13 ℃, but at -80 ℃ it is stable for at least 18 months. The reference interval for plasma total CoQ₁₀ in the New Zealand population is 0.47 - 1.80 µmol/L. There is no clinical requirement for stratification of the reference interval according to gender. Coenzyme Q₁₀ in human plasma is homeostatically controlled, varying little over a two month interval in healthy young males. Coenzyme Q₁₀ supplements have significantly different bioavailability, with the median increase in plasma CoQ₁₀ ranging from 0.14 to 0.59 µmol/L for seven different supplement brands. There is a large inter-individual variation in CoQ₁₀ absorption, and hence plasma concentrations should be monitored during supplementation. A plateau in CoQ₁₀ absorption, from a single dose, at approximately 200 mg suggests that the maximum dose ingested at one time should be 200 mg or less. Q-Gel capsules containing 30 mg of CoQ₁₀ are twice as effective at raising blood CoQ₁₀ as 100 mg capsules. Plasma CoQ₁₀ in patients with chronic heart failure are significantly lowered by approximately 33% when these patients receive Atorvastatin for six weeks. The absolute decrease in CoQ₁₀ showed a significant correlation with worsening endothelial function (r = + 0.548, p = 0.011). Coenzyme Q₉ was shown to be present in human plasma with a reference interval of 8.8 - 47.0 nmol/L.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.